Furocoumarin Derivatives from Radix Angelicae Dahuricae and Their Effects on RXRα Transcriptional Regulation

A novel furocoumarin derivative named oxyalloimperatorin (1), together with seventeen furocoumarins 2–18 were isolated from the radix of Angelica dahurica. The chemical structure of new metabolite was characterized by analysis of IR, NMR, and HR-ESI-MS spectroscopic data. Among the isolated compounds, 13, 16, and 18 (each at 20 μM) could significantly promote the gene transcriptional function of nuclear receptor RXRα. While 7–9, 13, 14, and the new structure 1 (each at 20 μM) showed significant reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid. The findings indicated that these furocoumarin skeleton derivatives might hold beneficial effects on many intractable diseases, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα.


Introduction
Radix Angelicae Dahuricae, the root of Angelica dahurica (Fisch. ex Hoffm) Benth, et Hook. f. var.formosana (Boiss.) Shan et Yuan, has been widely used as a traditional medicine in China for the treatment of toothache, headache, cough, asthma, coryza, etc. [1]. Previous phytochemical investigation revealed that the root of A. dahurica possesses various chemical composition including volatile oil, coumarins and glycosides. In them, coumarins were the most important major components with many remarkable activities, such as anti-inflammation [2], anti-bacteria [3], and lipogenetic inhibition [4]. In addition, many studies have been concerned about the anticancer effect of coumarins from A. dahurica [5][6][7][8].
The retinoid X receptor-α (RXRα) is a member of the nuclear receptor superfamily of ligandactivated transcription factors and an obligatory heterodimer partner for many nuclear receptors such as the peroxisome proliferator-actived receptor (PPAR), the retinoic acid receptor (RAR), and the liver X receptor (LXR) [9]. It plays key roles in various biological processes including cancer, diabetes, obesity, and atherosclerosis, and both agonist and antagonist of RXRα have been revealed to exert beneficial effects in such diseases [10][11][12]. In recent years, more and more studies have been focused on screening small molecules with regulatory function to RXRα from nature [13][14][15]. The specific aim of our present study was to identify novel naturally occurring metabolites with regulatory effects on RXRα gene transcriptional activation. Several furocoumarins were isolated from the radix of A. dahuricae and their transcriptional activities were examined by reporter gene assay.
The above findings indicate that these furocoumarin skeleton derivatives might have useful impact on many intractable diseases, such as cancer and metabolic diseases, due to their potential effects on regulating the transcriptional activation function of RXRα. Interestingly, compound 13 showed not only weakly increased the reporter transcriptional activation of RXRα but it also reduced the transactivity of RXRα induced by 9-cis-RA. One of the possible reasons could be that 13 and 9-cis-RA bind competitively with RXRα when they were simultaneously added to cells. Because of the transactive effect of 13 was rather weaker than that of 9-cis-RA (Figure 2), the transactivity of RXRα induced by 9-cis-RA was therefore decreased. However, this speculation and whether all the candidate compounds could bind to RXRα to regulate its transcriptional expression or not needs to be confirmed by further study.

General
High-performance liquid chromatography (HPLC)-grade solvents were purchased from Merck KGaA (Darmstadt, Germany). Analytical reagents were obtained from Sinopharm Chemical Reagent co., Ltd (Shanghai, China). Silica gel (200-300 mesh) used in column chromatography and TLC plates were bought from Qingdao Haiyang Chemical Co., Ltd (Qingdao, China). YMC gel ODS-A was purchased from YMC co., Ltd (Allentown, USA). NMR spectra were recorded on a Bruker Avance 400 spectrometer using tetramethylsilane as the internal reference. HR-ESI-MS were recorded on Bruker FT-MS. IR spectrum was carried on a Nicolet IR200 (Thermo Electron Corporation, U.S.A.). Rotation data was obtained from a 341 polarimeter (PerkinElmer Co. Ltd. U.S.A). The values of luciferases were measured on a 1420 VICTOR 3 TM V (PerkinElmer, Boston, MA, USA).

Materials
Radix

Cell Culture and Dual-Luciferase Reporter Gene Assay
Human embryonic kidney 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). The previous dual-luciferase reporter gene assay with some modification was used in the present study [14,27]. In brief, approximately 4 × 10 4 cells/well were seeded in 24-well plates. Cells were transfected with two plasmids, 30 ng pBind RXRα LBD and 60 ng pG5 luc using Lipofectamine 2000 (Invitrogen). After 24 h, cells were exposed to tested compounds for 12 h. Then, the cells were washed with PBS and lysed with passive lysis buffer (1 × PLB) on rocking platform for 15 min. The activities of Firefly luciferase and Rellina luciferase were examined according to the introduction of Dual-Luciferase Reporter Assay System Kit. Relative luciferase activities were obtained as the ratio between activities of Firefly luciferase and Rellina luciferase.

Statistical Analysis
The results were expressed as mean ± standard diviation (S.D.) from at least three independent experiments. Statistical significances were compared between two groups. Statistical analysis was performed with the Student's t-test. The value of P < 0.05 was considered statistically significant.

Conclusions
One novel furocoumarin derivative 1, together with seventeen furocoumarins 2-18 were isolated from the radix of Angelica dahurica. The chemical structure of new metabolite was characterized by analysis of IR, NMR, and HR-ESI-MS spectroscopic data. Among these furocoumarin skeleton derivatives, the new compound 1, and known compounds 7-9, 13, 14, 16 and 18 showed the potential activities in regulating transcriptional activation function of RXRα. These metabolites might show beneficial effects against intractable diseases with relation to RXRα, for example anti-cancer and antidiabetes. The various bioactivities of these metabolites and their molecular mechanism of action relating to nuclear receptor RXRα could be examined in the future study.